Particulate tissue graft with components of differing density and methods of making and using the same

ABSTRACT

Disclosed are tissue graft compositions made of particles having different densities, methods of making these compositions, and methods of using these compositions for promoting tissue restoration in a patient.

TECHNICAL FIELD OF THE INVENTION

The present invention is related to particulate tissue graftcompositions, methods of making them, and methods of using them torepair or restore tissues in a patient.

BACKGROUND

Tissue graft compositions made of devitalized native tissues have beenwidely used to repair or restore damaged, deformed, or missing tissuesin patients. Typically these compositions are made from theextracellular matrix of tissues, for example, the submucosa of theintestine (SIS), or urinary bladder (UBS), and the epithelial basementmembrane of, for example, the urinary bladder (UBM). Collectively thesecompositions are termed extracellular matrices or ECMs. SIS, UBS, andUBM are further described in U.S. Pat. Nos. 6,576,265, 6,579,538,5,573,784, 5,554,389, 4,956,178, and 4,902,508, each of which areincorporated by reference herein.

Briefly, SIS and UBS are made from gastrointestinal tissue and urinarybladder, respectively, by delaminating the submucosa from all otherlayers of the tissue source and retaining the submucosa to form a tissuegraft composition. UBM is made by delaminating the epithelial basementmembrane and, optionally, delaminating one or more of the deeper layersfrom the epithelial cells of the urinary bladder and retaining at leastthe epithelial basement membrane to form a tissue graft composition.Other tissues including, but not limited to skin and tissues of thegastrointestinal tract, e.g., esophagus, stomach, and intestine, mayalso be used to make an ECM including epithelial basement membrane bydelaminating the epithelial basement membrane from the epithelial cellsand, optionally, one or more of the deeper layers of the tissue source.Still other native ECMs may be made from connective tissues such asaponeurosis, tendon, ligament, smooth and skeletal muscle, andtreatment-site specific ECMs.

ECMs are used to restore, for example, epithelial tissues. Restorationof epithelial tissues requires restoration of the epithelium and theconnective tissue elements underlying the epithelial tissue. Epithelialrestoration occurs rapidly when epithelial basement membrane is present.However, restoration of the connective tissue elements is slower thanepithelial tissue restoration, prolonging the complete restoration ofdamaged epithelial tissues.

Each of the ECMs, such as UBS, UBM, and SIS, known in the prior art, areresorbed at the site of implantation in a patient. For example, a singlesheet of urinary bladder matrix (UBM) may resorb, for example, within aweek when implanted in a topical wound. FIGS. 1A-C depict the rapidresorption profile of a prior art ECM material 12 implanted at a tissuesite 10 in a patient. FIG. 1A shows the sheet of ECM 12 on day 0 ofimplantation. As shown, the ECM 12 fills the entire tissue site 10. Atday X after implantation, FIG. 1B shows that the ECM 12 has beenpartially resorbed at the tissue site 10 as shown by the reduced surfacearea of the ECM 12 as compared to the surface area of the ECM 12 in FIG.1A. At day Y after implantation, FIG. 1C shows that the ECM 12 has beencompletely resorbed by the tissue site 10 as no ECM 12 remains.

SUMMARY OF THE INVENTION

The invention disclosed herein has identified and solved the problem ofprior art ECM implants that are rapidly resorbed and are subsequently nolonger available to influence restoration and repair of damaged ormissing epithelial tissues and the underlying connective tissueelements. While the rapid initial resorption of prior art ECMs isbeneficial in providing an initial restoration response to the tissuesite in need of restoration or repair, the invention described herein isadvantageous because it provides, at the site in need of restoration orrepair, an ECM composition that takes longer to resorb than the priorart ECMs which prolongs the ECM's influence on the repair process at thesite of tissue injury. Accordingly, the invention disclosed herein isdirected to an ECM composition having at least one component thatresorbs quickly to provide a rapid initial repair response, but alsohaving at least one additional component that resorbs more slowly inorder to prolong the influence of the ECM composition combination on therestoration process at the site of tissue injury.

According to the invention, a preferred devitalized matrix for mammaliantissue repair or regeneration comprises at least a portion of amammalian epithelial basement membrane, preferably the entire epithelialbasement membrane, and the tunica propria that is immediately subjacentto the basement membrane. Devitalized matrices of the invention restoreor replace diseased, defective, or missing tissue when placed in contactwith host tissue. In a preferred embodiment, the invention comprises adevitalized matrix that is custom-shaped to conform to the diseased ordefective tissue. In a particular embodiment, the matrix comprises asheet of matrix derived from the urinary bladder, the intestine, or anyother mammalian epithelial tissue. In another embodiment, the matrix isinjectable by means of being transformed into a fine particulate,emulsion, gel or extract. A matrix of the invention may act as a carrierfor a pharmaceutical agent. A preferred application of the matrix of theinvention is the repair or restoration of cardiac tissue. In particular,a matrix or composition of the invention is useful to restore or replaceat least a portion of a cardiac valve, the interatrial septum, theinterventricular septum, or the myocardium. For the purposes of thisinvention, matrix and composition are interchangeable terms.

In one embodiment, the invention features a devitalized compositioncomprising epithelial basement membrane and tunica propria immediatelysubjacent to the basement membrane. The epithelial basement membrane andtunica propria immediately subjacent to the basement membrane aredelaminated from cells of a mammalian epithelium and abluminal portionsof the tunica propria. Mammalian epithelial tissue used in this aspectof the invention is preferably derived from urinary bladder, intestine,or any other mammalian epithelial tissue. Further embodiments feature acomposition shaped to conform to a diseased or defective cardiac valvesuch as at least a portion of a pulmonic valve, aortic valve, right orleft atrioventricular valve, or the myocardium.

In still another embodiment, the invention features a compositioncomprising epithelial basement membrane, tunica propria, and submucosa.The epithelial basement membrane and tunica propria are delaminated fromthe cells of an epithelium and from the tunica muscularis of a mammalianepithelial tissue.

In yet another embodiment, the invention features a compositioncomprising epithelial basement membrane, tunica propria, and smoothmuscle cells of the tunica muscularis, all delaminated from epithelialcells of a mammalian epithelium.

The composition, according to the invention, is not limited to merelythe embodiments enclosed. Rather, the composition, according to theinvention, comprises one or more layers of an epithelial tissue incombination with at least a portion, preferably the entire, intactepithelial basement membrane.

A preferred method of the invention comprises the step of contacting ahost tissue with a devitalized matrix derived from a mammal. Thedevitalized matrix comprises at least a portion of an epithelialbasement membrane and tunica propria immediately subjacent to thebasement membrane. In preferred embodiments, methods of the inventioncomprise inducing endogenous epithelial repair using tissue regenerativecompositions of the invention.

According to one aspect, the invention relates to compositions ofbioresorbable particles useful for tissue repair or restoration andmethods of using these compositions. The compositions includebioresorbable particles of differing density which resorb at differentrates, thereby providing the ability to adjust the length of timeprovided by the particles to effect repair as compared to compositionshaving materials of a single density.

According to one aspect, the invention includes tissue graftcompositions of bioresorbable particles. In one embodiment of theinvention, a mixture of bioresorbable particulate matter comprises afirst plurality of bioresorbable particles having a first density and afirst rate of resorption on implantation at a tissue site in the patientand a second plurality of particles having a second density and a secondrate of resorption on implantation at the tissue site in the patient.The second density is at least 150% of the first density. The secondrate of resorption is in the range of about 1 to about 10 times thefirst rate of resorption. The first plurality and second plurality ofbioresorbable particles promote tissue restoration and repair whenimplanted at a tissue site in a mammalian patient.

In one embodiment, the first plurality of particles comprises a firstbioresorbable material and the second plurality of particles comprises asecond bioresorbable material, whereas in another embodiment the firstplurality of particles and the second plurality of particles comprisethe same bioresorbable material. The first or second bioresorbablematerial may be selected from, for example, extracellular matrix,collagen, a bioresorbable polymer, or hyaluronic acid. The particles maybe in powder form, dispersed in a liquid or gel medium, or compressedinto a sheet form. The size of the particles may range from 1 μm to 1000μm. In a further embodiment, the particles may include one or morebioactive factors, pharmaceutical agents, or DNA vectors. The firstplurality of particles and the second plurality of particles may bepresent in a ratio of 1:1. The second density may be in the range from150% to 500% of the first density.

In yet another embodiment, the tissue graft composition includes a thirdplurality of particles having a third density that is more dense thanthe first and second densities and a third rate of resorption that isslower than the first and second rates of resorption on implantation atthe tissue site in a patient.

According to another aspect, the invention includes methods forpreparing mixtures of bioresorbable particles having differentdensities. For example, in one embodiment, a method for preparing amixture of bioresorbable particulate matter having differing densitiescomprises providing a first sheet of bioresorbable material having afirst density and a first rate of bioresorption and milling the sheetinto a first plurality of particles. A second sheet of a bioresorbablematerial having a second density and a second rate of bioresorption ismilled into a second plurality of particles. A predetermined quantity ofthe first plurality of particles is mixed with a predetermined quantityof the second plurality of particles, wherein the density of the secondplurality of particles is at least 150% of the density of the firstplurality of particles and wherein the first rate of resorption is atleast 1.5 times that of the second rate of resorption. The firstplurality of particles and the second plurality of particles are capableof promoting tissue restoration and repair when implanted at a tissuesite in a mammalian patient.

According to another embodiment of the invention, the second sheet maybe compressed or laminated prior to milling to increase its densityrelative to the first sheet. In an alternative embodiment, the firstsheet may be lyophilized prior to milling to reduce its density relativeto the second sheet.

According to another embodiment of the invention, the sheet may bemilled into particles by comminuting, grinding, chopping, blending,pulverizing, or mincing. In a further embodiment, the first or secondsheet is dried, hydrated or frozen prior to milling.

According to another embodiment of the invention, the bioresorbablematerial comprises extracellular matrix, hyaluronic acid or abioresorbable polymer. The hyaluronic acid or bioresorbable polymer maybe chemically synthesized or precipitated to form sheets of a firstdensity and sheets of a second density prior to milling.

According to yet another embodiment, the first sheet and the secondsheet may comprise the same bioresorbable material. For example, in oneembodiment the first and second sheet are extracellular matrix. Inanother embodiment, the first and second sheet are aponeurosis. In analternative embodiment, the first sheet and the second sheet comprisedifferent bioresorbable materials. For example, in one embodiment thefirst sheet comprises extracellular matrix and the second sheetcomprises hyaluronic acid. In another embodiment, the first or secondsheet further comprises one or more pharmaceutical agents, DNA vectors,or bioactive factors such as a growth factor.

In yet another embodiment, the second density of the second sheet is150%-500% of the first density.

In yet another embodiment, the particles of a first density and theparticles of a second density prepared by the method are compressed intoa sheet form.

In another embodiment of the invention, a method for preparing a mixtureof bioresorbable particulate matter having differing densities comprisesproviding a sheet of bioresorbable material of a first density andcompressing a portion of the sheet such that the portion comprises asecond density that is at least 150% or as much as 150%-500% of thefirst density. The sheet is milled into a plurality of particles. In afurther embodiment, the bioresorbable material is ECM.

In another aspect, the invention provides methods of promoting tissuerepair and restoration at an anatomical site in a patient. For example,according to one embodiment, a method of modulating the rate of tissuerepair at an anatomical site in a patient in need of repair orrestoration comprises administering to a patient at the site a mixturecomprising a first plurality of bioresorbable particles having a firstdensity and a second plurality of bioresorbable particles having asecond density at least 150% of the first density, wherein the secondplurality of bioresorbable particles are resorbed by the patient'stissue at a slower rate than the absorption of the first plurality ofbioresorbable particles, thereby prolonging the therapeutic effect ofthe second plurality of particles at the site. The first plurality andsecond plurality of bioresorbable particles promote tissue restorationand repair when implanted at the anatomical site in a mammalian patient.In a further embodiment, the mixture comprises a third plurality ofparticles having a third density.

In a further embodiment, the method also includes the steps of preparingthe mixture of bioresorbable particles which include the steps of (a)determining the optimal rate of absorption of a resorbable compositionfor repair or restoration of the site in the patient, (b) selecting thefirst plurality of bioresorbable particles to have a first density and afirst rate of absorption based on step (a) on implantation at a site inthe patient, and (c) selecting the second plurality of bioresorbableparticles to have a second density more dense than the density of thefirst plurality of particles and having a second rate of absorptionbased on step (a) and step (b) slower than the rate of absorption of thefirst plurality of bioresorbable particles on implantation at the sitein the patient.

According to one embodiment of the method, the bioresorbable particlesare extracellular matrix, collagen, a bioresorbable polymer, orhyaluronic acid. The bioresorbable particles may be delivered to thesite in the patient in powder form or in a liquid or gel medium. Thebioresorbable particles may also be injected at the site in the patient.The bioresorbable particles may contain one or more bioactive factors,DNA vectors, or pharmaceutical agents.

In another embodiment of the method, a mixture of bioresorbableparticles is administered to the patient via coating the mixture on amedical device prior to implantation of the medical device at the sitein the patient, whereas in an alternative embodiment, the mixture isadministered topically to the site. In a further embodiment, the mixtureis administered to the patient as a component of a bone substitutematerial.

According to a further embodiment of the method, the site in need ofrepair or restoration in the patient is a tendon, ligament, bone,kidney, liver, spleen, lymph node, urinary bladder, ureter, uterus,blood vessel, skin, breast, heart, or a body cavity. In one embodiment,the patient is a human.

According to another aspect, the invention includes an implant forimplanting in a patient comprising particles of extracellular matrix ofa first density and having a first rate of resorption in a patient andparticles of extracellular matrix of a second density and having asecond rate of resorption in a patient, where said particles arecompressed to form a three-dimensional object. The particles of thefirst density and the particles of the second density promote tissuerestoration and repair when implanted at a tissue site in a humanpatient.

According to one embodiment, the extracellular matrix is UBS, SIS, orUBM. In another embodiment, the particles of a first density are UBS andthe particles of a second density are UBM. In another embodiment of theimplant, the particles of the first density are more dense than theparticles of the second density and the particles of the first densityform a core of the implant and the particles of the second density formthe exterior surface of the implant.

In another aspect, the invention includes implanting the implant in apatient in need of tissue repair. In a further embodiment, the implantsite may be a tendon, ligament, bone, kidney, liver, spleen, lymph node,urinary bladder, ureter, uterus, blood vessel, skin, breast, heart, or abody cavity.

In yet another aspect, the invention includes a method of preparing amixture of particles of extracellular matrix which comprises preparing aplurality of particles of extracellular matrix having the same density,coating a first portion of the plurality of particles with a coatinghaving a first density and a first rate of absorption in a patient,coating a second portion of the plurality of particles with a coatinghaving a second density and a second rate of absorption in a patient,and mixing the first portion of the plurality of particles with thesecond portion of the plurality of particles. The plurality of particlespromote tissue restoration and repair when implanted at a tissue site ina mammalian patient. In one embodiment, the particles of extracellularmatrix include urinary basement membrane (UBM), the coating of the firstportion of the plurality of particles having a first density is UBM, andthe coating of the second portion of the plurality of particles having asecond density is small intestinal submucosa (SIS).

In yet another aspect, the invention is a composition of extracellularmatrix. The composition comprises a first sheet of extracellular matrix,a second sheet of extracellular matrix, and a first plurality ofextracellular matrix particles having a first density and a secondplurality of extracellular matrix particles having a second density atleast 150% of the first density, wherein the first plurality ofparticles and the second plurality of particles are contained betweenthe first sheet and the second sheet of extracellular matrix. The firstplurality and second plurality of extracellular matrix particles promotetissue restoration and repair when implanted at a tissue site in amammalian patient.

In a further embodiment, the composition includes a further plurality ofextracellular matrix particles having a density of either the firstdensity, the second density, or a third density, wherein the furtherplurality of particles are positioned between the surface of either thefirst sheet or second sheet of extracellular matrix and a third sheet ofextracellular matrix.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-C depict the bioresorption of a prior art ECM such as UBM, UBS,or SIS in a mammalian patient tissue. FIG. 1A depicts the ECM on Day 0at implantation. FIG. 1B depicts the ECM on Day X after implantationwhere the ECM has been partially resorbed by the patient tissue. FIG. 1Cdepicts the ECM on Day Y after implantation where the ECM has been fullyresorbed by the patient tissue.

FIGS. 2A-C depict the bioresorption of an exemplary particulate tissuegraft made up of two types of particles of different density accordingto one embodiment of the invention. The open circles (◯) represent alower density particle and the solid circles (●) represent a higherdensity particle. FIG. 2A depicts the particles on Day 0 atimplantation. FIG. 2B depicts the particles on Day X after implantationwhere the lower density particles have been partially resorbed by thepatient tissue, as evidenced by their decrease in size. FIG. 2C depictsthe particles on Day Y after implantation where the lower densityparticles have been fully resorbed by the patient tissue, and the higherdensity particles have not yet been fully resorbed by the patienttissue.

FIG. 3 depicts an exemplary composition of ECM particles according toone embodiment of the invention where the particles are of two differentdensities and the stages of an exemplary method of making thecomposition according to one embodiment of the invention. Two sheets ofECM are shown, one having density A and the other having density B. Thesheets are comminuted and a portion of A particles and B particles aremixed together.

FIG. 4 depicts an exemplary composition of ECM particles according toone embodiment of the invention where the particles are of two differentdensities and the steps of an exemplary method of making the compositionaccording to one embodiment of the invention. A sheet of ECM is treatedso that a portion of the sheet has a density A and a second portion ofthe sheet has a density B. The sheet is then comminuted to make acomposition of particles of two different densities.

FIG. 5 depicts an exemplary composition of ECM particles according toone embodiment of the invention where the particles have a density A, B,or C and the steps of an exemplary method of making the compositionaccording to one embodiment of the invention. A first sheet A of ECMhaving density A is adhered to a second sheet B of ECM having density B.The sheet is then comminuted to create a composition of particles havingdensity A and particles having density B and composite particles ofdensity C, the composite particles having components of both sheets Aand B.

FIG. 6 depicts an exemplary composition of ECM particles according toone embodiment of the invention where the particles have a core particleof the same density, but are coated with different materials havingdifferent densities A, B, and C. Accordingly, the coated particles havediffering densities A, B, and C.

FIG. 7 depicts an exemplary ECM composition according to one embodimentof the invention where ECM particles having densities A, B, and C areadhered to the surface of a sheet of ECM.

FIG. 8A depicts an exemplary ECM composition according to one embodimentof the invention where ECM particles of a higher density B arecompressed together to form a core of particles of density B coated withparticles of lower density A or layered with a layer of particles ofdensity A to form a construct in the shape of a mammalian body part,here exemplified as a kidney.

FIG. 8B depicts an exemplary ECM composition according to one embodimentof the invention where ECM particles of a higher density B arecompressed together to form a core of particles of density B coated withparticles of lower density A or layered with a layer of particles ofdensity A to form a construct in the shape of a mammalian body part,here exemplified as a human nose.

FIG. 9 depicts an exemplary ECM composition according to one embodimentof the invention where a mixture of ECM particles of a higher density Band of a lower density A are compressed together to form athree-dimensional construct in the shape of a mammalian body part, hereexemplified as a human nose.

FIG. 10 depicts an exemplary ECM composition according to one embodimentof the invention where a mixture of ECM particles of a higher density Band of a lower density A are compressed together to form a sheet.

FIG. 11 depicts an exemplary multi-laminate ECM composition according toone embodiment of the invention where a mixture of ECM particles of ahigher density B and a lower density A are sandwiched between sheets ofECM.

DETAILED DESCRIPTION OF THE INVENTION

Extracellular matrix materials (ECMs) have been used to promote repairand restoration of damaged, deformed, injured, and even missing bodytissues in mammals, such as humans. For example, ECMs such as SIS or UBMare implanted at a treatment site to promote repair and restoration ofepithelial tissues and connective tissues, including connective tissuelayers associated with epithelial tissues. Tissue sites in need ofrepair or restoration include, but are not limited to tendons,ligaments, breast, bone, kidney, liver, spleen, lymph nodes, urinarybladder, ureter, uterus, blood vessels, intestine, stomach, skin, heart,or a body cavity. “Restore,” “restored,” “restoring,” or “restoration”means that tissue function and structure is substantially returned toits original condition by the patient's endogenous tissue repairmechanisms in combination with the tissue graft composition.

ECMs such as SIS, UBS, and UBM are rapidly resorbed by host tissues inwhich the ECM has been implanted. Once the material has been resorbed bythe host, the ECM no longer has an influence over tissue restoration andrepair at the site of implantation. As ECMs are resorbed, they releasebioactive components such as growth factors and other bioactivecomponents naturally present in the native ECM that influence tissuerestoration and repair at the site of implantation of the ECM. Once theECM has been fully resorbed, these growth factors and bioactivecomponents are no longer released by the ECM at the implantation site toinfluence tissue repair and restoration. In order to moderate theappearance of these factors and components contributed by ECMs duringrepair of tissues, disclosed herein are compositions of ECM particlesthat have resorption times that vary from the rapid resorption times ofknown native ECMs.

In one aspect, the invention relates to tissue graft compositions of ECMparticles where the particles vary in density. “Density” as used hereinrefers to mass per unit volume of the particle. According to oneembodiment of the invention, ECMs are manipulated to alter theirdensities and are then particularized. The particles, when implanted ina patient, resorb at rates related to their densities, with lowerdensity particles being resorbed by the patient at a faster rate thanmore dense particles. Lower density particles resorb more quickly thanhigher density particles, releasing growth factors and other bioactivecomponents quickly, thereby modulating an initial repair response at thetissue site. In contrast, higher density particles resorb slowly,releasing growth factors and other bioactive components more slowly,thereby maintaining the repair response at the site of implantationbeyond the initial repair response influenced by the growth factors andbioactive components released by the lower density particles.

According to the invention, having both lower and higher densityparticles as opposed to just higher density particles alone permits thereleased growth factors and other bioactive components provided by thelower density particles to modulate an initial rapid repair response atthe tissue site due to the rapid resorption of the low densityparticles. Ideally, such a rapid repair response cannot be provided bythe higher density particles alone because their resorption is slowerand therefore, the release of bioactive components from the materialthat encourages tissue repair and restoration is also slower.

Traditional ECMs have a rapid resorption time such that the ECMs may befully resorbed at the tissue implantation site before the tissue site iscompletely repaired. Increased density ECM particles or aggregates ofECM particles of varying densities prepared according to the inventionhave prolonged resorption times and can therefore have an extendedinfluence over tissue repair and restoration at the site of the injuryas compared to using traditional ECMs. In other words, tissue graftcompositions according to the invention allow the tissue site to repaircompletely or close to completely before the tissue graft composition isbioresorbed at the tissue site. According to the invention, compositionsof ECM particles provide a specific bioresorption profile tailored tothe restoration needs of a specific tissue site in a patient.

The invention as described herein has the advantage of reducing theamount of space required to accommodate an ECM tissue graft construct atan implantation site in a patient. For example, by increasing thedensity of a tissue graft construct according to the invention, more ECMmaterial can be included in a smaller volume. In addition, by increasingthe density, the ECM material according to the invention takes longer tobe resorbed at the implantation site in comparison to thicker, lessdense ECM materials in the patient, thereby exerting a prolongedinfluence on tissue repair and restoration at the implantation site inthe patient.

According to the invention, the rate of resorption of compositions canbe measured based on the number of days required for a certain volume ofECM to be resorbed by host tissue at the site of implantation. Forexample, a composition according to the invention, in one embodiment,includes a lower density particle having a volume of 100 μM² and adensity of X that takes 15 days to be resorbed and a higher densityparticle having a volume of 100 μM² and having a density of 2× thattakes 45 days to be resorbed. According to the invention, there is notnecessarily a direct linear relationship between density and resorptiontime. As shown by this example, a particle that is twice the density ofa first less dense particle may take longer than twice the resorptiontime of the first less dense particle to be resorbed.

As previously mentioned, ECMs known in the art have a rapidbioresorption rate when implanted at a tissue site in a patient. Incontrast to the rapid bioresorption profile demonstrated by known ECMs,compositions according to the invention have varied bioresorption timesbased on the density of particles in the tissue graft composition. Forexample, FIGS. 2A-C demonstrate the varied bioresorption profile of atissue graft composition of particles of differing density according tothe invention. FIG. 2A shows ECM particles 16 (of a higher density thanthe ECM shown in FIGS. 1A-C) and ECM particles 14 (of comparable densityto the ECM shown in FIGS. 1A-C) on day 0, the day of implantation at thetissue site. At day X after implantation, as shown in FIG. 2B, the lowdensity particles 14 have been partially resorbed as indicated by theirreduced size. The more dense particles 16 have also not yet been fullyresorbed. By day Y after implantation, as shown in FIG. 2C, the lowerdensity particles 14 have been fully resorbed, while the more denseparticles 16 have not, permitting the more dense particles to continueto promote tissue repair and restoration at the tissue site 10.

In one embodiment of the invention, a tissue graft composition ofparticles includes particles of a first density and particles of asecond, different density. In a further embodiment, the composition alsoincludes particles of a third density; particles of a third and fourthdensity; particles of a third, fourth, and fifth density; or particlesof a third, fourth, fifth, and sixth density, and so on. For example, inone embodiment, the particle composition has particles of two densities;in another, three densities; in yet another, four densities; in stillanother, five densities; and in yet another, six densities. According toone embodiment of the invention, the composition has particles of morethan six different densities. In yet another embodiment, a particle maybe a composite of more than one material, for example, a combination ofdifferent ECM types, each ECM type having a different density.

In one embodiment of the invention, the composition has particles of afirst density and particles of a second different density where theparticles of the second density are more dense than the particles of thefirst density. In a further embodiment, the particles of the seconddensity are at least about 150% of the density of the particles of thefirst density (i.e., 1.5 times more dense). This magnitude of differencein density is necessary such that the difference between the rate ofbioresorption of the particles of the first density and the rate ofbioresorption of the particles of the second density is large enough toallow the tissue being treated to be repaired before the composition isfully resorbed. Particles of a second density having a density less thanabout 150% of the first density do not have a bioresorption profilesignificantly different from the bioresorption profile of particles ofthe first density to allow the tissue site to be repaired before thecomposition is completely resorbed.

In a further embodiment, the composition further includes particles of athird density. Particles of the third density are more dense thanparticles of the second density; the particles of the second density aremore dense than particles of the first density. In a particularembodiment, the particles of the third density are at least about 150%of the density of the particles of the second density and the particlesof the second density are at least about 150% of the density of theparticles of the first density (i.e., 1.5 times more dense). Again, thismagnitude of difference in density is necessary such that the differencebetween the rates of bioresorption between the particles of the firstdensity, the second density, and the third density is large enough toallow the tissue being treated to be repaired before the composition isfully resorbed.

In a further embodiment, the particles of a second density that are moredense than particles of a first density have a resorption rate of about1 time, about 2 times, about 3 times, about 4 times, about 5 times,about 6 times, about 7 times, about 8 times, about 9 times, or about 10times, for example, the rate of resorption of the particles of a first,less density.

In a further embodiment, the particles of a second density are at leastabout 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, or 5times more dense than the density of the particles of a first density.In another embodiment, the particles of a third density are at leastabout 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, or 5times more dense than the particles of a second density.

Preferably, the second density particles are at least about 150-500% ofthe density of the lower first density particles, more preferably about200-400% of the density of the lower first density particles, and mostpreferably about 200-300% of the density of the lower first densityparticles.

The particles according to one embodiment of the invention areextracellular matrix particles (ECM). According to the invention, ECMfrom which the particles of differing density are made may be derivedfrom native tissues including, but not limited to submucosa, dermis,epithelial basement membrane, aponeurosis, tendon, ligament, smooth andskeletal muscle, and treatment site-specific ECM. The native tissuesource may be porcine, bovine, or ovine, for example. The native tissuesource may be allogenic, autogenic, or xenogenic to the patient. Forexample, if the patient is a human, the native tissue source is fromanother human (allogenic) or from the patient (autogenic).Alternatively, if the patient is a human, the native tissue is from anon-human species (xenogenic).

ECM particles according to the invention have densities ranging fromabout 150 mg/cm³ to about 1800 mg/cm³, more preferably from about 150mg/cm³ to about 1200 mg/cm³, and most preferably from about 150 mg/cm³to about 600 mg/cm³.

In another embodiment of the invention, particles of the invention arecomposed of, for example, collagen, i.e., type I, II, III and/or IVcollagen; a bioresorbable polymer such as poly (L) lactic acid, poly (D)lactic acid, polyglycolic acid, polydioxanone, or tyrosinepolycarbonate, and/or hyaluronic acid. Collagen particles according tothe invention have densities ranging from about 150 mg/cm³ to about 1800mg/cm³, more preferably from about 150 mg/cm³ to about 1200 mg/cm³, andmost preferably from about 150 mg/cm³ to about 600 mg/cm³. Bioresorbablepolymer particles according to the invention have densities ranging fromabout 150 mg/cm³ to about 1800 mg/cm³, more preferably from about 150mg/cm³ to about 1200 mg/cm³, and most preferably from about 150 mg/cm³to about 600 mg/cm³.

According to one embodiment of the invention, bioactive materials suchas growth factors, DNA, or other proteins are added to particles ofbioresorbable polymers for release as the polymers are resorbed in thepatient. In another embodiment, bioactive materials such as growthfactors, DNA, or other proteins are added to ECM compositions of theinvention for release as the ECM is resorbed in the patient.

In one embodiment according to the invention, all particles of the firstdensity are made from a first starting material and all particles of thesecond density are made from the same first starting material. Forexample, in one embodiment, all particles of the first density are madeof an ECM material having a first density and all particles of thesecond density are particles of the same ECM material, but manipulatedto have a second, different density. For example, the particles of afirst density and the particles of the second density are each UBMparticles. In another embodiment, all particles of the first density area bioresorbable polymer and all particles of the second density are thesame bioresorbable polymer. For example, the particles of the firstdensity and the particles of the second density are poly (L) lacticacid.

In another embodiment, all particles of the first density are made froma first material and all particles of the second density are made from asecond material. For example, all particles of the first density aremade from a first ECM material and all particles of the second densityare made from a second ECM material. For example, in one embodiment, theparticles of the first density are made of UBM while particles of thesecond density are made from SIS. In another embodiment, all particlesof the first density are made from an ECM material while all particlesof the second density are made from a bioresorbable polymer. In anotherembodiment, the first material is UBM, while the second material isaponeurosis. In another embodiment, the first material is UBM, while thesecond material is liver basement membrane (LBM). In yet anotherembodiment, the first material is UBM, while the second material ishyaluronic acid. In an even further embodiment, the first material isUBM, while the second material is poly(lactic-co-glycolicacid) (PLGA)

Particles according to one embodiment of the invention range from about0.1 μM to about 2000 μM. More preferably, the particles range in sizefrom about 1 μM-about 2000 μM, even more preferably from about 1μM-about 1000 μM, and most preferably from about 50 μM-about 750 μM inwidth, for example, diameter.

According to one embodiment of the invention, the mixture of differingdensity particles is maintained in a powder form, while in anotherembodiment, the mixture of particles is maintained in a gel or liquidcarrier for injection at a tissue site in a patient. Examples ofsuitable carriers include but are not limited to hyaluronic acid,gelatin, lecithin, collagen gel, and saline.

In a further embodiment, particles according to the invention containgrowth factors and/or other biologically active molecules. For example,particles such as ECM may naturally contain biologically activemolecules that are released at the site of implantation as the ECM isbioresorbed by the patient. Alternatively, biologically active moleculesmay be added to the composition of particles to be delivered to the siteof implantation with the particles. In yet another embodiment, particlesaccording to the invention are coated with biologically activemolecules. In yet another embodiment, particles of bioresorbablepolymers are synthesized and formed into particles in the presence ofbiologically active molecules so that the biologically active moleculesare contained within the bioresorbable polymer particle.

Examples of biologically active molecules include, but are not limitedto epidermal growth factor, TGF-alpha, TGF-beta, fibroblast growthfactor, platelet derived growth factor, vascular endothelial growthfactor, insulin-like growth factor, keratinocyte growth factor, and bonemorphogenic protein to name a few, as well as pharmaceutical agents, andDNA vectors.

According to the invention, particles release biologically activemolecules in a time dependent fashion. For example, given that lowerdensity particles are bioresorbed by patient tissue at a more rapid ratethan higher density particles, biologically active molecules, such asgrowth factors, are released from lower density particles relativelyquickly after implantation as compared to their release from higherdensity particles. Higher density particles bioresorb more slowly andtherefore release biologically active molecules at the tissue site overa longer period of time than the lower density particles, therebyexposing the tissue site to particular biologically active molecules fora longer period of time than if only lower density particles arepresent.

According to another aspect, the invention relates to methods of makingmixtures of particles where a first type of particle has a first densityand a second type of particle has a second density.

According to one embodiment of the method of making mixtures ofparticles of the invention, sheets of ECM are comminuted to create amixture of particles of differing density. For example, as shown in FIG.3, a mixture 24 of A particles 14 and B particles 16 where A particles14 are less dense than B particles 16 is made from two ECM sheets 20, 22of differing densities. A Sheet 20 is comminuted to form A particles bychopping, cutting, pulverizing, milling, or grinding A Sheet 20 with asuitable device that forms particles such as a blender, a hammer mill, aknife mill, a centrifugal mill, a die and press system, or a rollercrusher, for example. B Sheet 22 is comminuted to form B particles 16 bychopping, cutting, crushing, pulverizing, milling, mincing, or grindingB Sheet 22 with a suitable device to form particles 16. A particles 14and B particles 16 have size ranges and density ranges as describedabove.

To form the mixture, a predetermined number of A particles 14 is thenmeasured and mixed with a predetermined number of B particles 16 to forma composite mixture 24 of A particles 14 and B particles 16, where theconcentration of A particles 14 to B particles 16 is predetermined basedon a particular desired resorption profile of the mixture at a tissuesite in a patient. For example, for a tissue site that takes a long timeto repair, such as a joint, fewer less dense A particles 14 are selectedcompared to the number of higher density B particles 16 in order toprovide a prolonged resorption of ECM at the tissue site. A particles 14may be selected at a ratio of (A:B) 1:1, 1:2, 1:3, 1:4:2:3, 3:4, 2:1,3:1, 3:2, 4:3, or 4:1 B particles 16, for example, depending on theparticular tissue site to be treated and the desired resorption profileof the particles for treating that tissue site.

According to a further embodiment of the invention, a mixture ofparticles includes three types of particles, each type of a differingdensity. For example, the mixture includes low density particles,intermediate density particles, and higher density particles. Forexample, according to the invention, a sheet of a third density (C) iscomminuted to form particles of a third density C, higher than thedensity of particles formed from A sheet 20 and B sheet 22. Apredetermined number of particles of the third density are measured outand added to the mixture of A particles 14 and B particles 16. Particlesof a third density maybe be selected at a ratio of (A:B:C) 1:1:1, 1:1:2,1:1:3, 1:1:4, 1:2:1, 1:2:2, 1:2:3, 1:2:4, 1:3:1, 1:3:2, 1:3:3, 1:3:4,1:4:1, 1:4:2, 1:4:3, 1:4:4, 2:1:1, 2:1:2, 2:1:3, 2:1:4, 2:2:1, 2:2:3,2:3:1, 2:3:2, 2:3:3, 2:3:4, 2:4:1, 2:4:3, 3:1:1, 3:1:2: 3:1:3, 3:1:4,3:2:1, 3:2:2, 3:2:3, 3:2:4, 3:3:1, 3:3:2, 3:3:4, 3:4:1, 3:4:2, 3:4:3, or3:4:4, for example, depending on the particular tissue site to betreated and the predetermined resorption profile of the particles fortreating the tissue site.

For example, in one embodiment, a composition of the invention has twoparts lower density particles to 1 part higher density particles (2:1)and is used for wound treatment, i.e., of the skin and skin adnexa. Thehigher concentration of lower density particles, when applied to awound, provides a rapid influx of bioactive components to activate therestoration and repair process as the lower density particles arebioresorbed, followed by a longer term release of bioactive componentsover time at the implantation site as the higher density particlesresorb slowly over time. Additionally, specific bioactive components maybe added to the lower or higher density particles to tailor the releaseof bioactive components to the stage of restoration and repair at thetissue site.

In another embodiment, a composition of the invention has 1 part lowerdensity particles to four parts higher density particles (1:4) or 1 partlowest density particles, 2 parts lower density particles, and 2 partshighest density particles, (1:2:2) and is used for tendon or ligamentrepair. Restoration and repair of tendons and ligaments takes longerthan at body sites that are typically more vascular and more cellular.Accordingly, the 1:4 or 1:2:2 density profile composition provides alonger resorption profile of the composition for restoring or repairingtendons and ligaments as compared to traditional ECMs.

In yet another embodiment, a composition of the invention has 2 partslower density particles to 3 parts higher density particles to (2:3) andis used for scar revision or plastic surgery applications to promote arapid early tissue restoration response while also having prolongedbioactive component release at the site to help prevent hypertrophicscar formation over a longer time.

In yet another embodiment, a composition of the invention has one partlower density particles to 2 parts higher density particles (1:2) and isused for applications involving soft tissue augmentation. In a furtherembodiment, a composition of the invention has one part lower densityparticles to 3 parts higher density particles (1:3) and is used forophthalmological applications.

According to the invention, sheets of ECM 20, 22 can be treated to altertheir densities prior to being comminuted. The density of a sheet of ECMcan be increased by mechanically compressing the material, dehydratingthe material, vacuuming the material, cross-linking the material, orlyophilizing the material, for example. The density of a sheet of ECMcan be decreased by hydration, expanding the pore structure undervacuum, mechanically expanding the material, or freezing the material inwater, for example.

In another embodiment of the method of making a mixture of particleshaving a first density A and particles having a second density B,particulate matter from a sheet of ECM having areas of density A anddensity B are removed from the sheet of ECM and mixed together. Forexample, as shown in FIG. 4, sheet 30 is partially compressed to form asheet 30 having an area A 31 of a first lower density and area B 32 of asecond higher density. The sheet 30 is then milled to produce particles33 from area A 31 having a lower density A and particles 34 from area B32 having a higher density B.

According to one embodiment of the invention, the portion of sheet 30that is compressed relative to the portion that is not compressed orless compressed is determined based on the predetermined bioresorptionprofile for the particles at a given tissue site in a patient.Accordingly, for example, if a 1:1 ratio of A:B (low density:highdensity) particles is sought to achieve the desired bioresorptionprofile, one half of the sheet is compressed to the desired densityprior to comminuting the sheet into a mixture of particles of A and Bdensities. In one embodiment, the size of sheet 30 is selected prior tocompression of a portion of the sheet 30 and the whole sheet 30 iscomminuted after compression. Alternatively, in another embodiment, aportion of the sheet 30 is compressed after which a portion of sheet 30is cut away from the whole of sheet 30 with the cut-away portion beingcomminuted.

In a further embodiment, a portion of the sheet 30 is compressed to havea third density C. The sheet 30 is then milled to produce a mixture ofparticles having a density A, a density B, and the third density C. Thethird density may be higher than A or higher than A and B.

Another embodiment of the method of making a mixture of particles havingdiffering densities is shown in FIG. 5. According to one embodiment, asheet 40 of ECM of a first density A is joined with a sheet 41 of ECM ofa second density B to form a composite sheet 45. The composite sheet 45is then comminuted to create a mixture of particles 46 having differentdensities. For example, when composite sheet 45 is comminuted, themixture includes A particles 42 of low density derived from sheet A, Bparticles 43 of a higher density derived from sheet B, and C particles44 of an intermediate density formed from the interfacing area 47 of theA sheet 40 and the B sheet 41.

A further embodiment of the method of making a mixture of particleshaving different densities is shown in FIG. 6. According to thisembodiment, particles 65 have a core 61 and a coating 62. The core 61has a density X and may be coated with any of a variety of coatings 63,64, 66 having a variety of densities. For example, some particles 65have cores 61 coated with a first material 66 having a first density toproduce particles having a density A, while other particles 65 havecores 61 that are coated with a second material having a second density63 to produce particles having a density B, while yet other particles 65have cores 61 coated with a third material having a third density 64 toproduce particles having a third density C. Coating the core 61 can beperformed, for example, by precipitating the coating onto a particlefrom a solution, by a roll coater, or by a granulator. Materials forcoatings may include, for example, an ECM, a gel made from an ECM suchas UBM gel or SIS gel, PLGA, hyaluronic acid, or collagen, for example.The core 61 of particles 65 may include, for example, ECMs as describedherein.

In another aspect of the invention, mixtures of comminuted particles ofdiffering density are coated on devices such as synthetic or naturalwound repair matrices for implantation into a patient. For example, asshown in FIG. 7, a mixture of A lower density particles 52 and B higherdensity particles 53 is applied to a sheet 50 of ECM. The particles 52,53 are adhered to the sheet by compression or use of an adhesive, forexample. Accordingly, when the sheet 50 is implanted at a tissue site ina patient, the particles 52, 53 provide more surface area to contactwith the tissue site in the patient. The A particles 52 and B particles53 are more dense in one embodiment than the ECM sheet 50 so that afterthe sheet 50 has been fully resorbed, the tissue continues to repair inthe presence of particles 52 and 53. The remaining particles 52, 53allow the tissue to repair before the particles 52, 53 of thecomposition are completely resorbed.

In a further embodiment, mixtures of comminuted particles of differingdensity coated on a device such as a synthetic or natural wound repairmatrix is implanted in a patient for use as an adhesion barrier. Forexample, as shown in FIG. 7, a matrix in sheet form 50, for example, iscoated on one side with particles 52, 53 of differing density accordingto the invention and is implanted in a patient. Any adhesions forming atthe implant site adhere to the particulate side of the matrix and as theparticles 52, 53 are resorbed, the adhesions no longer remain attachedto the matrix, but to the particles and the adhesions are subsequentlyresorbed. In an alternative embodiment, particles of differing densityaccording to the invention are sandwiched between a first matrix sheetof a first heavier density and a second matrix sheet of a lighterdensity to form an implant. The implant is implanted at a site in thepatient with the more dense matrix sheet implanted against the tissuerequiring repair and the less dense sheet facing outward into the bodyof the patient. As adhesions form on the outward facing less densesheet, the less dense sheet is resorbed allowing any adhesions to becomeunattached from the more dense portion of the implant, the adhesionsthen being subsequently resorbed by the patient along with the lessdense sheet of matrix.

In one embodiment, the mixture of particles 52, 53 is adhered to onesurface of the sheet 50, while in another embodiment the mixture ofparticles 52, 53 is adhered to all surfaces of the sheet 50. Forexample, the sheet 50 bioresorbs in the patient within 60 days, thelower density A particles bioresorb in 7 days, and the higher density Bparticles bioresorb in the patient within 30 days.

In a further embodiment, a mixture of comminuted particles according tothe invention is coated on a surgical medical device prior to thedevices' implantation at a tissue site in a patient. For example, amixture of lower density A particles and higher density B particles arecoated on a medical implant such as, but not limited to, a stent, acardiac occluder, suture material, or a mesh. The particles are adheredto the device by, for example, a chemical bond, a peptide linker, abioresorbable adhesive, such as cyanoacrylate or fibrin glue, or bymechanical means such as lyophilization of the particles on the deviceor compressing the particles onto the device. Once the device isimplanted at the tissue site in the patient, the lower density Aparticles bioresorb quickly providing an initial restoration response atthe tissue site in the patient, while the higher density B particles arenot fully bioresorbed until the tissue site is completely repaired. Forexample, the lower density A particles are bioresorbed by the patientwithin, for example, 7 days, while the higher density B particles arebioresorbed by the patient within, for example, 30 days.

In another aspect, the invention includes aggregates of particles ofdiffering density which form an implant with portions of differingdensities for implantation at a tissue site in a patient. According tothe invention, as shown in FIGS. 8A and 8B, high density particles B arecompressed to form a shape replicating a body part or portion of a bodypart in a mammal, for example, a human, and form the core 71 a, b of animplant 70, 72. Lower density particles A are then compressed or coatedaround the 71 a, b core of B particles to form the peripheral layer ofthe implant 70, 72. As shown in FIG. 8A, the A and B particles form anovoid shape implant 70 to be used in replacement or repair, for example,of a kidney or liver. As shown in FIG. 8B, the peripheral A and core Bparticles form roughly a triangular shape implant 72 to be used inreplacement or repair, for example, of a nose.

The implant 70, 72 is implanted into a patient at a site requiringrestoration or repair, for example. Upon implantation, the peripheral Aparticles having a lower density provide an immediate repair andrestoration response at the tissue site in the patient. After theperipheral A particles rapidly bioresorb, the higher density core 71 a,b B particles continue to provide a repair and restoration response atthe tissue site in the patient over a longer period of time than theperipheral A particles alone. The core 71 a, b B particles are typicallynot fully resorbed by the patient until the tissue site is repaired. Forexample, the peripheral A particles of lower density are bioresorbed bythe patient within, for example, 14 days, while the core B particles ofhigher density are bioresorbed by the patient within, for example, 60days.

In a further embodiment, as shown in FIG. 9, an implant 74 can be formedby compressing a mixture of lower density A particles 76 with higherdensity B particles 78. The lower density particles 76 resorb at afaster rate which create channels in the implant and provide space forbody tissue to grow into the implant, while the higher density particlesresorb more slowly, providing structural support for the construct astissue restoration and repair occurs and releasing bioactive componentsover time as the higher density particles resorb. The implant 74 can becompressed into any number of three dimensional shapes such as a kidney,spleen, nose, ear, liver, bone, or other body organ.

In another embodiment of the invention, a three dimensional construct ofcompressed particles of varying density is made to mimic naturalbiologic tissue densities. For example, in one embodiment, a constructis made with denser particulate matter on one side, of a sheet forexample, and more dense particulate matter is placed on the other side.Such a construct can be used at a bone-cartilage interface where themore dense portion is inserted into the bone defect while the less denseportion is positioned in the cartilage defect. The slower resorbing,higher density particulate remodels according to the slower rate of bonegeneration resulting in more dense tissue restoration consistent withbone, while the less dense portion remodels more quickly and creates aless dense tissue restoration consistent with cartilage. The inventionalso contemplates three dimensional constructs of compressed particulatehaving different density regions that are designed, for example, tomimic the native density variations of a spinal disc or themuscle-tendon interface.

In a further embodiment, as shown in FIG. 10, a sheet 80 is formed bycompressing a mixture of lower density A particles 81 with higherdensity B particles 82. The sheet 80 can be formed, for example, byhydrating the particles and compressing the particles together byapplication of a mechanical force or vacuum to form a cake or wafer-likeconstruct. The sheet 80 can be used to treat topical wounds, forexample, of the skin, but can also be used to treat other areas of thebody such as a hernia, intestine, bowel, blood vessels, the heart, orany other organ, by cutting the sheet to size and/or bending or wrappingthe sheet to fit the implant location. The sheet 80, with its differingdensity particles, will break down on implantation as a result ofbioresorption of the lower density particles, providing greater surfacearea for tissue restoration and repair at the site of implantation. Thesheet form 82 has the benefit of being easy to apply to the implantsite, but also has the ability to provide a prolonged restorationresponse at the implant site as the particles 81, 82 are resorbed atdifferent rates, in contrast to sheets made of only one density ofmaterial.

In a further embodiment, as shown in FIG. 11, mixtures of particles 91of differing densities, i.e., A particles of a first density 92 and Bparticles of a second density 93, are sandwiched between sheets 94 of anECM. For example, a powder form mixture of particles of a first densityA 92 and a second density B 93 is sandwiched between sheets 94 of UBM orSIS to form a multi-laminate construct 95 of loose particles 91 andsheets 94. Any number of alternating layers of sheets 94 and the mixture91 of particles 92, 93 can be constructed according to the invention.For example, in one embodiment, a first sheet 94A is covered with theparticle mixture 91; the particle mixture 91 is covered with a secondsheet 94B; the second sheet 94B is covered with the particle mixture 91;and a third sheet 94C applied to form asheet-particle-sheet-particle-sheet multi-laminate construct 95. Thesheets of multi-laminate construct 95, when implanted at a site in apatient, provide a microarchitecture for tissue restoration at the sitewhile the particles 92, 93 breaking down at varying rates provide acontinuous supply of bioactive factors to assist in restoration at theimplant site.

In a further embodiment, particles of a lower density A and particles ofa higher density B can be combined with a bone substitute material suchas beta-tricalcium phosphate (βTCP), hydroxyapatite, sintered bovinebone, sintered porcine bone, demineralized allograft bone, ormineralized allograft bone, for example. In an alternative embodiment,the bone particles and the differing density ECM particles of theinvention can be mixed together without an adhesive, binder, or anyother additive. The particles are coated on and/or injected into thebone substitute material. When the bone substitute material is implantedat the tissue site in the patient, the bone substitute material providesa rigid form to stabilize the defect while the lower density particlespromote rapid tissue repair at the site. Over time, the higher densityparticles sustain tissue repair at the site of implantation as they arebioresorbed at a slower rate than the lower density particles. Thehigher density particles are typically not fully bioresorbed until thetissue site is repaired.

According to another embodiment of the invention, a mixture of particleshaving particles of a lower density A and particles of a higher densityB are provided in powder form. A powder has particles of 1-1000 μm. Thepowder can be sprinkled topically on a tissue site in a patient, forexample, a cut, scrape, incision, or puncture of the skin, or while atissue site is exposed during surgery, the powder form can be sprinkledat the tissue site. In another embodiment, the differing densityparticles are administered via inhalation, for example, through use ofan inhaler such as those used to administered asthma treatments.

In an alternative embodiment, a mixture of particles of a lower densityA and particles of a higher density B are maintained in a liquid, gel orpaste medium. The liquid or gel is injected via a syringe at a tissuesite in a patient. Alternatively, the liquid or gel is maintained withina tube and is squeezed out of the tube and applied to the tissue site.In a further embodiment, the powder, liquid, gel, or paste compositionincludes particles of a third density.

In another aspect, the invention encompasses methods for modulating therate of tissue restoration at a tissue site in a patient in need ofrepair or restoration. The method requires administering to a site in apatient a mixture that includes at least two types of particles, eachtype having a different density, one type of particle being more densethan the other type of particle. The differing density particles areselected to form the mixture based on their respective bioresorptionprofiles, i.e., the higher density particles are selected because theytake longer to be resorbed at the tissue site than the particles of alower density and the lower density particles are selected because theywill be resorbed quickly at the tissue site. According to the invention,a physician particularly selects the mixture to have particles withparticular rates of bioresorption tailored for the particular tissuesite to be repaired or restored. The mixture is administered to thepatient topically, via injection to the tissue site, or it may bedelivered to the tissue site surgically, for example.

Tissue sites treatable with compositions according to the inventioninclude, but are not limited to head and neck structures, jointsincluding synovium, joint capsule, labrum, intraarticular ligaments andcartilage, intervertebral discs, pelvic floor, tendon, bone, ligament,kidney, spleen, liver, muscle tissue, urinary bladder, ureter, uterus,intestine, pancreas, blood vessel, skin and skin adnexa, heart includingintracardiac structures, nose, ear, breast, or a body cavity.

EXAMPLES

1. Tissue Restoration of a Chronic Skin Ulcer

A mixture of ECM particles is made by taking 60 mg of particles of UBMof size 50-1000 μm and having a density of approximately 0.05 g/cm³ and20 mg of particles of UBM of size 50-500 μm and having a density ofapproximately 0.25 g/cm³, for a 3:1 ratio of particles based on weight.The mixture is applied topically to a chronic skin ulcer. Within 10days, the 0.05 g/cm³ particles are resorbed and tissue restoration isobserved at the wound site. Within 30 days, the 0.25 g/cm³ particles areresorbed and tissue at the wound site is fully restored.

2. Tissue Restoration of a Kidney

A mixture of ECM particles is prepared for implantation in a kidney at asite from which a tumor has been excised. A mixture of ECM particles ismade by taking 200 mg of particles of UBM of size 500-1000 μm and havinga density of approximately 0.10 g/cm³ and 1000 mg of particles of UBM ofsize 50-500 μm and having a density of approximately 0.35 g/cm³, for a1:5 ratio of particles based on weight. The mixture is surgicallyimplanted in the patient via a syringe. Within 20 days, the 0.10 g/cm³particles have resorbed and tissue restoration is observed at the woundsite via magnetic resonance imaging. Within 60 days, the 0.35 g/cm³particles are resorbed and tissue at the wound site is fully restored asviewed via magnetic resonance imaging.

What is claimed is:
 1. A method for preparing a mixture of bioresorbableparticulate matter, comprising: (a) providing a first sheet ofbioresorbable material comprising a native extracellular matrixcomprising epithelial basement membrane and tunica propria and having afirst density and a first rate of resorption, and, milling said firstsheet into a first plurality of bioresorbable particles; (b) providing asecond sheet of a bioresorbable material comprising anotherextracellular matrix different than the extracellular matrix material ofthe first sheet and having a second density and a second rate ofresorption, and, milling said second sheet into a second plurality ofbioresorbable particles; (c) providing a predetermined quantity of saidfirst plurality of bioresorbable particles and a predetermined quantityof said second plurality of bioresorbable particles; and (d) mixing saidpredetermined quantity of said first plurality of bioresorbableparticles with a predetermined quantity of said second plurality ofbioresorbable particles, wherein the second density is 150%-500% of thefirst density.
 2. The method of claim 1, further comprising compressingor laminating the second sheet prior to milling to increase its densityrelative to the first sheet.
 3. The method of claim 1 further comprisinglyophilizing the first sheet prior to milling to reduce its densityrelative to the second sheet.
 4. A method for preparing a mixture ofbioresorbable particulate matter having differing densities comprising:providing a first sheet of bioresorbable material comprising a nativeextracellular matrix comprising epithelial basement membrane and tunicapropria and having a first density, and, compressing a portion of saidsheet such that said portion comprises a second density; providing asecond sheet of bioresorbable material comprising a native extracellularmatrix; joining said first sheet to said second sheet to form amultilaminate sheet, and milling said multilaminate sheet into aplurality of bioresorbable particles, wherein said mixture ofbioresorbable particles promotes tissue restoration and repair whenimplanted at a tissue site in a mammalian patient, wherein the seconddensity of the first sheet is at least 150% of the first density.
 5. Amethod of preparing a mixture of bioresorbable extracellular matrixparticles, comprising: preparing a plurality of bioresorbable particlesfrom a native extracellular matrix comprising epithelial basementmembrane and tunica propria each having the same density; dividing theplurality of bioresorbable particles into a first group and a secondgroup; coating the first group of said plurality of bioresorbableparticles with a coating comprising a material selected from the groupconsisting of an extracellular matrix, polyglycolic acid, hyaluronicacid, collagen, or combinations thereof, said coating having a firstdensity; coating the second group of said plurality of bioresorbableparticles with a coating comprising a material selected from the groupconsisting of an extracellular matrix, polyglycolic acid, hyaluronicacid, collagen, or combinations thereof, said coating having a seconddensity; mixing said first group of bioresorbable particles with saidsecond group of bioresorbable particles to form a mixture ofbioresorbable particles, wherein said mixture of bioresorbable particlespromotes tissue restoration and repair when implanted at a tissue sitein a mammalian patient, and wherein the second density is 150%-500% ofthe first density.
 6. The method of claim 4 wherein said first sheet ofbioresorbable material and said second sheet of bioresorbable materialcomprises non-cross-linked extracellular matrix.
 7. The method of claim5 wherein said native extracellular matrix is non-cross-linked.
 8. Themethod of claim 1 wherein said first sheet and said second sheetcomprise non-cross-linked extracellular matrix.
 9. The method of claim 1further comprising providing a biologically active component selectedfrom the group consisting of epidermal growth factor, TGF-alpha,TGF-beta, fibroblast growth factor, platelet derived growth factor,vascular endothelial growth factor, insulin-like growth factor,keratinocyte growth factor, bone morphogenic protein, pharmaceuticalagents, DNA vectors and combinations thereof.
 10. The method of claim 1wherein the density of the second plurality of bioresorbable particlesis at least 150% of the density of the first plurality of bioresorbableparticles and wherein the first rate of resorption is at least 1.5 timesthat of the second rate of resorption.